The present invention relates to the production of pyrimidines, purines and derivatives thereof e.g. deoxyribonucleosides, using genetically modified cells comprising novel DNA constructs.
Thymidine is useful as a pharmaceutical intermediate, particularly for the chemical synthesis of azidothymidine (xe2x80x9cAZT,xe2x80x9d sold under the trademark ZIDOVUDINE). Although ZIDOVUDINE-type AZT was one of the first therapies developed for HIV/AIDS, it continues to have important and expanded use (Langreth, R., The Wall Street Journal, Nov. 21, 1995, pp B12). ZIDOVUDINE-type AZT is valuable particularly when used in combination therapies such as a combination with lamivudine (also known as 3TC), sold under the trademark EPIVIR. This lamvudine and 3TC combination is sold under the trademark COMBIVIR. Although the HIV virus can mutate to form resistance to either AZT or 3TC, COMBIVIR-type nucleotide-analog combination is particularly effective because the reverse transcriptase apparently cannot be resistant to both nucleoside analogues at the same time (Larder, B. A. et al., Science 269: 696-699, 1995). ZIDOVUDINE-type AZT is also useful in conjunction with HIV protease inhibitor type drugs (Waldholz, M., The Wall Street Journal, Jan. 30, 1996, pp B1), and in the treatment of HIV infected pregnant women in order to reduce the frequency of infection of the fetus at birth. In 1997 about 600,000 children died of AIDS contracted from their mothers at birth. ZIDOVUDINE-type AZT taken for several months prior to birth can reduce the transmission of the virus to infants by two-thirds. Thymidine produced by chemical synthesis used in the manufacture of AZT is a very significant cost.
In U.S. Pat. No. 5,213,972 (McCandliss and Anderson, hereinafter xe2x80x9cthe ""972 patentxe2x80x9d), the entire contents of which are incorporated herein by reference and to which the reader is specifically referred, a process for the production of pyrimidine deoxyribonucleoside (PdN) is disclosed (see in particular examples 7 to 14 of the ""972 patent). A replicatable microorganism comprising and expressing a DNA sequence encoding a pyrimidine deoxyribonucleotide phosphohydrolase that converts a PdN monophosphate to a pyrimidine deoxyribonucleoside is taught. More particularly, McCandliss and Anderson, supra, describe a fermentation method that can be used to produce thymidine that involves the expression of deoxythymidylate phosphohydrolase (dTMPase) from the Bacillus bacteriophage PBS1. This type of enzyme has been found in nature expressed by bacteriophages that do not contain thymidine in their DNA, but instead incorporates compounds like deoxyuridine or hydroxymethyl-deoxyuridine.
In the thymidine fermentation described in the ""972 patent, the enzymes that degrade thymidine (thymidine phosphorylase and uridine phosphorylase) have been removed by mutation so that thymidine accumulates. Thus, the use of the dTMPase enzyme helps create the pathway to allow thymidine synthesis. An expression of dTMPase alone, however, may not assure a commercially viable level of thymidine production. Accordingly, there is a continuing need to enhance the production of thymidine by cells expressing dTMPase in order to make thymidine production by fermentation commercially viable, by lowering the production cost relative to the current chemical synthesis methods.
The biochemical pathway for pyrimidine deoxynucleotide production, for example, in E. coli is highly regulated at the levels of transcription and translation as well as at the protein level by mechanisms including attenuation, feedback inhibition and enzyme activation. Neuhard, J. and R. A. Kelln, Biosynthesis and Conversion of Pyrimidines, Chapter 35 [In] Neidhardt, F. C. et al. [eds] xe2x80x9cEscherichia coli and Salmonella Cellular and Molecular Biologyxe2x80x9d, Second Edition, Vol. I, pp580-599, ASM Press, Washington D.C., 1996. The expression of dTMPase and elimination of thymidine breakdown by mutations in the deoA (thymidine phosphorylase), udp (uridine phosphorylase) and tdk (thymidine kinase) genes and therefore resulting expression products results in thymidine synthesis in E. coli but not at a commerically viable level.
The biosynthesis of purines and pyrimidines involves a common step of reducing a ribonucleoside diphosphate (in some species triphosphate) to its corresponding deoxy analog. In the overall process the reduction of the ribose moiety to 2-deoxyribose requires a pair of hydrogen atoms which are ultimately donated by NADPH and H+. However, the immediate electron donor is not NADPH but the reduced form of a heat stable protein called thioredoxin or glutaredoxin and at least one other unidentified source since the E. coli ribonucleotide reductase system still works in trxA (thioredoxin) grx (glutaredoxin) double mutants (Neuhard and Kelln, supra). The reducing equivalents of the reduced thioredoxin are transferred to ribonucleoside diphosphate reductase which carries out the reduction process. Manipulation of, for example, this step could prove useful in improving the commerical production of purine and pyrimidine deoxynucleosides.
It is an object of the present invention to provide novel DNA constructs e.g. vectors and genetically modified microorganisms comprising said vectors particularly for use in the production of recoverable amounts, especially commercially useful amounts, of pyrimidine and purine deoxynucleosides.
It is also an object of the present invention to provide processes which represent an improvement over McCandliss and Anderson described supra.
In accordance with one aspect of the present invention there is provided a DNA construct comprising a transcriptional unit which comprises a ribonucleotide reductase gene and a thioredoxin gene or a uridine kinase gene and/or a dCTP deaminase gene.
In one embodiment the DNA construct comprises a transcriptional unit which comprises a ribonucleotide reductase gene and a thioredoxin gene.
In another embodiment the DNA construct comprises a transcriptional unit which comprises a uridine kinase gene and/or a dCTP deaminase gene.
Preferably the DNA construct comprises a transcriptional unit which comprises a uridine kinase gene and a dCTP deaminase gene.
Most preferably the DNA construct comprises a transcriptional unit which comprises a ribonucleotide reductase gene and a thioredoxin gene and a uridine kinase gene and a dCTP deaminase gene.
In accordance with another aspect of the present invention there is provided a modified host cell comprising a DNA construct according to the invention.
In accordance with yet another aspect of the present invention there is provided a culture medium comprising the modified host cells of the invention and processes for the production of a purine or pyrimidine, for example thymidine comprising the use of said modified host cells.
In one embodiment the host cells comprise a DNA construct which construct comprises a transcription DNA unit (e.g. operon) which unit comprises DNA sequences encoding for ribonucleotide reductase and thioredoxin in which said reductase preferably displays less sensitivity to allosteric inhibition than a wild type host cell equivalent or counterpart wherein said cell further comprises one or more of the following features:
(a) a transcription unit (e.g. operon), preferably located on said DNA construct, comprising DNA sequences encoding for (and preferably heterologous with respect to host cell equivalent) thymidylate synthase;
(b) a transcription unit (e.g. operon), preferably located on said DNA construct, comprising DNA sequences encoding for uridine kinase and preferably dCTP deaminase; and
(c) repressed or absent Uracil DNA glycosylase activity.
In another embodiment the DNA construct for use in the production of recoverable amounts of pyrimidine and derivatives thereof, in particular pyrimidine deoxyribonucleosides such as thymidine, comprises a transcription unit (e.g. operon) which unit comprises (preferably heterologous) DNA sequences encoding for uridine kinase and/or dCTP deaminase.
Genetically modified host cells comprising and expressing the construct and culture medium comprising the modified host cells are also provided.
This aspect is based, in part, on the observation that host cells comprising DNA encoding for uridine kinase and/or dCTP deaminase, optionally togather with additional genes as suggested in U.S. Pat. No. 5,213,972 required for thymidine production, lead to a significant improvement in thymidine production.
The respective aspects of the present invention disclose for the first time a plurality of advances on the teaching of U.S. Pat. No. 5,213,972 to provide improved DNA constructs and host cells comprising the constructs for use in the commercial production of pyrimidine deoxyribonucleosides, particularly thymidine.
Other objects, features and advantages of the present invention will become apparent from the following description. It should be understood, however, that these represent preferred embodiments of the invention and are by way of illustration only. Various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art.
The construct of the present invention may be chromosomal or more preferably extra-chromosomal e.g. located on a vector.
Vectors of the present invention include plasmid, virus, transposons, minichromosome or phage, preferably plasmid. The vector comprising the transcription unit may be introduced into the host cell according to any convenient method known to those skilled in the art, e.g. P1 transduction, electroporation or transformation. Suitable host cells useful in the present invention include eukaryotes and prokaryotes (e.g. Bacterium). Prokaryotes include E. coli, Salmonella, Pseudomonas, Bacillus, strains and mutants thereof. E. coli is preferred due to the large amount of information, genetic tools and mutant alleles that are available. It is particularly preferred that a method of transduction is available for the host cell of choice to enable mutations to be readily moved from one host cell to another and facilitate genetic mutation of the host without requiring direct mutation whenever a new mutation is desired.
The present inventors have found that the use of bacteriophage T4 nrdA, nrdB and nrdC genes are particularly useful for encoding the reductase and thioredoxin in E. coli. See Sjxc3x6berg, B. M. et al., EMBO J., 5:2031-2036 (1986); Tseng, M.-J., et al., J. Biol. Chem. 263:16242-16251 (1988); and LeMaster, D. M., J. Virol. 59:759-760 (1986). More specifically, a very significant improvement in E. coli thymidine production was achieved through the cloning and expression of the T4 bacteriophage nrdA and nrdB genes coding for ribonucleotide reductase together with T4 nrdC coding for thioredoxin since the T4 ribonucleotide reductase cannot use E. coli thioredoxin. The T4-coded ribonucleotide reductase was found to be relatively insensitive to control by allosteric inhibition in vitro compared to the E. coli enzyme (Berglund, O., J. Biol. Chem. 247:7276-7281, 1972). For example, unlike the E. coli enzyme (Berglund, O., J. Biol. Chem. 247: 270-7275, 1972) the T4 ribonucleotide reductase is not inhibited by dATP, but actually stimulated by DATP and ATP (Berglund, O., J. Biol. Chem. 247:7276-7281, 1972).
DNA sequences encoding for the ribonucleotide reductase (e.g. nrdA and nrdB genes) and thioredoxin (e.g. nrdC gene) are preferably heterologous with respect to host cell DNA and preferably derived from T phage (preferably E. coli T bacteriophage), particularly T xe2x80x9cevenxe2x80x9d phages e.g. T2, T4 or T6. See Campbell, A. M., Bacteriophages, Chapter 123, In Neidherdt, supra; and Mathews, C. K. et al. (eds.) Bacteriaophage T4, American Society of Microbiology, Washington, D.C., 1983. The term xe2x80x9cderived fromxe2x80x9d is intended to define not only a source in the sense of its physical origin but also to define material which has structural and/or functional characteristics which correspond to material originating from the reference source.
Another useful feature of the T even phage enzyme is its substrate specificity. The normal E. coli ribonucleotide reductase uses UDP as a substrate only poorly since the Km for UDP is about 10 fold higher for UDP than CDP (Neuhard and Kelln, supra). However, the T4 enzyme has only a two-fold difference in Km (Berglund, O., J. Biol. Chem. 247: 7276-7281, 1972) between CDP and UDP substrates allowing two routes to dUTP synthesis. Although there have been attempts to obtain functional expression of T4 ribonucleotide reductase in E. coli, previous efforts were only successful in expressing the components separately and could demonstrate activity only by mixing in vitro (Tseng, M.-J., P. He, J. M. Hilfinger, and G. R. Greenberg, J. Bacteriol. 172: 6323-6332, 1990). Whilst not being bound by theory, the inventors believe that perhaps due to the lack of the usual pattern of feedback inhibition, expression of T4 ribonucleotide reductase in E. coli is lethal and it must be carefully conditionally expressed. Further envisaged are genes that encode precursor forms of the reductase and/or thioredoxin which are processed to produce a mature form. Such processing may proceed via various intermediate forms.
Vectors of the present invention preferably comprise a regulatory element (e.g. promoter such as lambda PL, operator, activator, repressor such as lambda repressor, particularly a temperature sensitive variant, and/or enhancer), appropriate termination sequences initiation sequences and ribosome binding sites. The vector may further comprise a selectable marker. Alternatively, regulatory elements (particularly lambda repressor) may be located on the host cell chromosome. It is preferred that nrdA and nrdB are arranged in the vector downstream (in terms of reading frame) from nrdC. In particular, it is preferred that nrdB is arranged downstream from nrdA. Thus a most preferred arrangement is a vector comprising an operon comprising nrdCAB.
The T4 ribonucleotide reductase is not devoid of feedback-control in vivo (J. Ji, R. G. Sargent, and C. K. Mathews, J.Biol.Chem. 266: 16289-16292, 1991; and Berglund supra). To promote ribonucleoside diphosphate reduction further e.g. for thymidine production, the gene coding for the regulatory subunit, nrdA, may be modified by, for example, a mutational approach to create an enzyme capable of increased thymidine production due to e.g. a reduced sensitivity to allosteric inhibition for example inhibition by the enzyme""s immediate product or inhibition by a product resulting from a downstream event.
In order to construct T4 nrdA mutants, site-directed mutagenesis may be used to modify or change (e.g. substitute) gene bases encoding amino acids suspected to alter e.g. dTTP binding site involved in allosteric regulation. Analysis of the amino acid sequence of T4 ribonucleotide reductase revealed a segment that appears to fit well with a postulated consensus sequence thought to be involved in dTTP binding (E. M. McIntosh and R. H. Haynes, Mol. Cell. Biol. 6:1711-1721, 1986). Several changes may be made in this region of the T4 ribonucleotide reductase using oligonucleotide-directed mutagenesis. The general approach may be modelled after the effort of More et al. (More, J. T., J. M. Ciesla, L.-M. Changchien, G. F. Maley and F. Maley, Biochemistry 33: 2104-2112, 1994) to reduce the dTTP binding of deoxycytidylate deaminase. One mutation, 79Ala to Ile, in the T4 nrdA appeared to be very useful. For example, the thymidine productivity of strains containing the 79Ala to lie mutant in T4 nrdA evaluated by a shake flask fermentation method was significantly increased. As demonstrated below, the present inventors achieved at least 25% increase over the parent strain without this single change.
Although the 79Ala to Ile is one successful example, those skilled in the art will now realize that many other amino acid changes to this region are now possible to obtain the desired effect, that being to putatively disrupt dTTP binding, but not disrupt the enzyme""s basic functionality. For example, substitution of 79Ala with other amino acids displaying similar side chains to lie (e.g. leucine, valine) may be utilized. Modifications of position 79 in conjunction with other modifications (e.g. mutations) within the postulated consensus region are also envisaged. Deletion of one or more amino acid positions in the consensus region and introduction of synthetic DNA into the region are other approaches available to those skilled in the art.
In another aspect of the present invention there is provided a host cell comprising a construct which construct (e.g. vector) comprises a transcriptional unit comprising DNA sequences encoding for heterologous ribonucleotide reductase and thioredoxin which reductase is less sensitive to allosteric inhibition than the wild type host cell equivalent or counterpart. It will be apparent to those skilled in the art that determining the relative sensitivity of a candidate heterologous reductase to allosteric inhibition compared to the wild type host cell equivalent is a matter of routine experimentation and observation.
Transcription units comprising the DNA sequences e.g. nrdA, nrdB and nrdC genes are preferably operons wherein the nrd genes are arranged in tandem. This permits transcription of these genes as a single mRNA transcript. In order to minimize unproductive energy expenditure by the host cell and further to minimize plasmid size, it is preferred that the operon contains only genetic sequences required in the encoding of reductase and thioredoxin (including any regulatory or control elements). This may necessitate the removal of superfluous DNA (for example, the unusual intron in the phage T4 nrdB gene, Sjoberg, B-M., et al EMBO J.5: 2031-2036, 1986).
In other preferred embodiments, vectors of the present invention for use in for example the production of thymidine further comprise DNA sequences encoding for thymidylate synthase (e.g. the td gene). See e.g. Chu, F. K. et al., Proc. Natl. Acad. Sci. USA 81:3049-3053 (1984); Chu, F. K. et al., J. Bacteriol. 169:4368-4375 (1987). The purpose of using this enzyme is to improve control over the levels of deoxyuridine produced and in particular the relative impurity level of deoxyuridine relative to thymidine. The dTMPase enzyme is not completely specific for dTMP. With a higher Km than for dTMP, the PBS1 dTMPase will also utilize dUMP as substrate to produce deoxyuridine (Price, A. R., Methods in Enzymol. 51: 285-290, 1978). Deoxyuridine creates a significant problem for thymidine purification. Therefore, one way to reduce deoxyuridine production is to efficiently convert dUMP to dTMP by increasing the level or effectiveness of thymidylate synthase such that the internal concentration of dUMP always remains very low.
The thymidylate synthase gene (td) may be heterologous with respect to the host cell and it is preferred that td is derived from (in the sense defined supra) T bacteriophage, e.g. T xe2x80x9cevenxe2x80x9d phage and in particular T4 phage td. Although td may be located in its own transcription unit, it is preferred that td is located in the same transcription unit e.g. operon as nrd genes. Moreover, it is preferred that td is located in the same operon downstream (in terms of reading frame) from the nrd genes.
McCandliss and Anderson, supra, amplified the E. coli thymidylate synthase gene in plasmids pCG138 and pCG148 (see Table 5, of the ""972 patent) and it was found to be partially effective in reducing deoxyuridine. The T4 thymidylate synthase is much more effective which is surprising in light of the fact that the E. coli enzyme is not thought to be controlled by any type of allosteric regulation (Neuhard and Kelln, supra). The E. coli enzyme Km for dUMP, 4 xcexcM (Wahba, A. J. and M. Friedkin, J. Biol. Chem. 237: 3794-3801), and the T4 enzyme Km for dUMP, 2.73 xcexcM (Maley, F., L. LaPat-Polasko, V. Frasca and G. F. Maley, Functional domains In T4 Thymidylate Synthase as probed by site-directed mutagenesis, Chapter 29 [In] Karam, J. D. [ed] xe2x80x9cMolecular Biology of Bacteriophage T4xe2x80x9d, American society for Microbiology, Washington, D.C., 1994, pp 322-325), are similar and cannot explain the large difference in effectiveness. Whilst not being bound by theory, the inventors believe that the E. coli thyA has an internal transcription termination sequence derived from an upstream gene that could be effecting the expression level in plasmid clones (Bell-Penderson, D, J. L. Galloway Salvo, and M. Belfort, J. Bacteriol. 173:1193-1200, 1991).
In other preferred embodiments, host cells of the present invention, particularly for use in the commercial production of pyrimidine deoxyribonucleosides e.g. thymidine comprise a transcription unit (e.g. operon) which unit comprises DNA sequences e.g. udk gene encoding for uridine kinase and preferably DNA sequences e.g. dcd gene encoding for dCTP deaminase. See e.g. Wang, L. and B. Weiss, J. Bacteriol. 174:5647-5653 (1992); and Neuhard, J. and L. Tarpxc3x8, J. Bacteriol. 175: 5742-5743.
The construct of this aspect of the invention may additionally comprise a transcription unit encoding for ribonucleotide reductase (nrdA and nrdB) and the thioredoxin (nrdC), or precursor forms thereof which are preferably heterologous with respect to host cell DNA and preferably derived from E. coli bacteriophage, particularly T xe2x80x9cevenxe2x80x9d phages e.g. T2,T4 or T6.
Uridine kinase produces UMP and CMP from uridine and cytidine using GTP (or dGTP) as the phosphate donor. The reaction is inhibited by UTP and CTP (J. Neuhard and R. D. Kelln, Biosynthesis and Conversions of Pyrimidines, Chapter 35 [in] F. C. Neidhart et al. [ed], xe2x80x9cEscherichia Coli and Salmonella Cellular and Molecular Biologyxe2x80x9d, Second Edition, ASM press, Washington D.C.). The present inventors have found that the use of uridine kinase particularly together with dCTP deaminase leads to a marked improvement in the production of thymidine by host cells incorporating these changes together with the teachings of ""972 outlined supra. This observation is quite unexpected since uridine kinase, on the basis of current information, has no direct role in pyrimidine de novo biosynthesis, moreover that its use would be beneficial in commercial processes for the production of pyrimidine deoxyribonucleosides. It is preferred that udk and dcd genes are arranged in tandem in the same operon. Further envisaged are genes that encode precursor forms of the udk and dcd gene which are processed to produce a mature form. Such processing may proceed via various intermediate forms. The udk and dcd genes may be introduced into the construct (e.g. vector) from any suitable source by methods well known to those skilled in the art for example P1 transduction, electroporation or transformation.
The enzyme uracil DNA glycosylase, encoded by the ung gene, is responsible for degrading DNA that has uracil incorporated in place of thymine. Where host cells of the present invention are used in the commercial production of e.g. thymidine, the internal cellular concentration of dTTP may be lowered as a result of the utilization of dTMP (a precursor of dTTP) in the production of thymidine. Accordingly, the present inventors have recognized that there Is potentially a greater propensity for uracil incorporation into the host DNA which may be lethal to a wild type host due to the uracil DNA glycosylase activity causing too many single stranded breaks in the host cell DNA. Thus, host cells useful In the present invention may further display repressed (compared to the unmodified cell) or no uracil DNA glycosylase activity. This repression or absence may be achieved through various ways apparent to those skilled in the art. For example, antagonism (either total or partial) of the ung gene expression products is one such approach by introducing an antagonist of the functional enzyme (or precursor thereof) into the host cell. Other approaches include manipulating ung gene expression by e.g. modifying regulatory elements of ung gene expression or introducing mutations into the ung gene itself such that ung gene product expression displays little or no uracil DNA glycosylase protein and/or activity. Another approach is to delete the ung gene (or functionally critical parts thereof) from host cell DNA. The absence or low level of uracil DNA glycosylase activity may be a feature of the host cell without the need for further manipulation.
In preferred embodiments of the present invention, each of the advances taught herein are incorporated into a host cell. The nrd, td, udk and dcd genes may be located on separate constructs but it is preferred that they are all located on the same construct e.g. vector. Thus in a particularly preferred embodiment of the present invention, a modified host cell is provided in which the cell comprises a DNA construct (e.g. vector) comprising a transcription DNA unit (e.g. operon) which unit comprises DNA sequences encoding for preferably a T even phage, e.g. T4) a modified ribonucleotide reductase and thioredoxin in which said reductase preferably displays less sensitivity to allosteric inhibition than wild type host cell equivalent or counterpart wherein said construct further comprises:
(a) a transcription unit (e.g. operon) encoding for (preferably heterologous with respect to host cell equivalent or counterpart) thymidylate synthase and;
(b) a transcription unit (e.g. operon), encoding for uridine kinase and preferably dCTP deaminase; and in which the host cell displays repressed or absent uracil DNA glycosylase activity.
Host cells modified according to the present invention are particularly useful in the commercial production of pyrimidine deoxynucleosides. In a particularly advantageous use of the present invention, E. coli host cells comprising (harboring) a plasmid modified according to the present invention (particularly in conjunction with the teachings of the ""972 patent) may be used in the commercial production of thymidine. Thus, host cells modified according to the present invention may further comprise dTMPase derived from e.g. PBS1 and the mutations taught in the ""972 patent, e.g. deoA, tdk-1 and udp-1.
Generally, a fermentation method is employed which involves submerging the cells in a culture medium contained within a suitable vessel. Following culturing under appropriate conditions, produced thymidine is harvested and purified (enriched), if necessary, to pharmaceutical grade according to standard protocols. The purified thymidine may then be used in the production of medicaments, e.g. pharmaceutical compositions such as AZT.